1. Field of the Invention
The present invention relates to a process for inducing a transcriptionally active chromosome (i.e., a "lampbrush" chromosome) from condensed chromatin or nuclei.
2. Description of the Related Art
More than 100 years ago, Flemming described giant chromosomes in the oocyte nucleus or germinal vesicle (GV) of the salamander Ambystoma mexicanum. His initial brief observations were soon followed by detailed investigations that established the existence of giant chromosomes in GVs of many animals, both vertebrate and invertebrate. Ruckert named them lampbrush chromosomes (LBCs) because of their fancied resemblance to the brushes used for cleaning kerosene lamp chimneys.
Lampbrush chromosomes of amphibian oocytes are the largest known chromosomes, rivaled only by the giant polytene chromosomes of Drosophila and other flies. They are characterized by the presence of hundreds or thousands of transcriptionally-active regions that loop out laterally from the main axis of the chromosome. Each loop consists of one or a small number of active genes. Striking variations in the morphology and/or molecular composition of individual loops and of other landmarks along the main axis (e.g., centromeres, telomeres, axial granules, and the like) make it possible to map these chromosomes at the gene level by conventional light microscopy, using combinations of morphology, antibody binding, and in situ nucleic acid hybridization.
Although lampbrush chromosomes are found in a variety of organisms, they are conspicuously absent from many others or are not analyzable in some cases because of the small size of germinal vesicles in some organisms. For instance, it is still debatable whether typical lampbrush chromosomes occur in mammalian oocytes, including human oocytes, but even if they do, the germinal vesicle is too small to analyze such chromosomes by the techniques of the prior art.
The overall organization and functional significance of lampbrush chromosomes have been the subject of extensive experimentation and speculation (reviewed in Callan, 1986; Davidson, 1986). Because LBCs occur in oocytes during the growth period, they are in an arrested diplotene phase of meiosis I with homologous chromosomes held together at one or more chiasmata. Each homologue has a DAPI-positive axis of chromomeres, which correspond to transcriptionally-inactive regions where sisters are intimately associated. Numerous pairs of loops extend laterally from the chromomere axis; these consist of transcriptionally-active regions where sisters are completely independent of one another. Even at the light microscope level of analysis, loops are not of uniform thickness, but contain of one or more "thin-to-thick" regions, which correspond to transcription units. The electron microscope shows that the "thin-to-thick" morphology reflects the increasing length of nascent RNP fibrils along the transcription unit in the direction of transcription. This structure of LBCs reflects their transcriptionally active state.
Despite a wealth of detailed morphological and molecular information on lampbrush chromosomes, fundamental questions remain about their structure and especially about the significance of their highly active transcription. At one time their structure was regarded as unusual, but the discovery of looped chromatin domains in somatic nuclei (Paulson and Laemmli, 1977; Saitoh and Laemmli, 1993; Yokota et al., 1995) suggests that the lampbrush condition is a good model for chromosomes in general. However, the cis and trans factors which cause a condensed chromosome to assume the lampbrush condition had not been identified.
These and other questions about LBCs would be easier to address in a system in which the lampbrush state could be artificially induced and manipulated. In the present application, we disclose that condensed chromatin injected into a heterologous germinal vesicle gives rise within hours to distinctive transcriptionally-active LBCs. This system will be useful in assessing the relative importance of cis and trans acting factors in establishing the morphological and molecular attributes of LBCs. It should also permit analysis of transcriptionally-active chromosomes from organisms whose oocytes cannot be handled by current techniques or do not go through a typical lampbrush stage. Furthermore, LBCs from interphase nuclei exhibit a pattern of loops useful for cytogenetic analysis with higher resolution than interphase chromosomes painted by fluorescent in situ hybridization. Such loop patterns allow mapping, essentially at the single gene level.